Valves and pumps for microfluidic systems and method for making microfluidic systems

ABSTRACT

The present invention relates to microfluidic systems, including valves and pumps for microfluidic systems. The valves of the invention include check valves such as diaphragm valves and flap valves. Other valves of the invention include one-use valves. The pumps of the present invention include a reservoir and at least two check valves. The reservoir may be of variable volume. The present invention also relates to a flexible microfluidic system. The present invention additionally relates to a method of making microfluidic systems including those of the present invention. The method includes forming a microfluidic system on a master, connecting a support to the microfluidic system and removing the microfluidic system from the master. The support may remain connected to the microfluidic system or the microfluidic system may be transferred to another substrate. The present invention further relates to a method of manipulating a flow of a fluid in a microfluidic system. This method includes initiating fluid flow in a first direction and inhibiting fluid flow in a second direction and may be practiced with the valves of the present invention.

[0001] This patent application is a continuation application of U.S.patent application Ser. No. 10/041,718, filed Jan. 8, 2002, which isincorporated herein by reference, and claims priority to U.S. PatentApplication No. 60/260,221, filed Jan. 8, 2001, U.S. Patent ApplicationNo. 60/327,430, filed Oct. 5, 2001 and U.S. Patent Application No.60/331,856, filed Nov. 20, 2001.

[0002] This invention was sponsored by NSF Grant Nos. ECS-9729405,ECS-0004030, MRSEC DMR-9809363 and AFOSR/SPAWAR Grant No.N66001-98-1-8915. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to microfluidic systems, includingflexible microfluidic systems and valves and pumps for microfluidicsystems. The present invention also relates to a method of making amicrofluidic system suitable for use with a polymeric material.

BACKGROUND OF THE INVENTION

[0004] Microfluidic systems are flow systems miniaturized to dimensionsas small as a few micrometers (μm). Such systems present challenges inboth their design and manufacture. For example, at the level ofminiaturization of typical microfluidic systems, normal fluid flowprinciples may be less significant than surface tension.

[0005] Recent developments in microfluidic systems have been motivatedin large part by the possibility of fabricating compact, integrateddevices for analytical functions such as genomic analysis, diagnosis andsensing.

SUMMARY OF THE INVENTION

[0006] According to one embodiment of the present invention, amicrofluidic system is provided including a fluid path, an inlet and anoutlet to the fluid path, and a first closing member disposed along thefluid path between the inlet and the outlet. In this embodiment of theinvention, the fluid path has a cross-sectional dimension of less thanabout 500 μm.

[0007] According to another embodiment of the present invention, a valvehaving an open position and a closed position is provided. The valveincludes a fluid path and an inlet and an outlet to the fluid path. Aflexible diaphragm having an opening is disposed along the fluid pathbetween the inlet and the outlet to the fluid path. In this embodimentof the invention, a seat is constructed and arranged such that, when thevalve is in the closed position, the seat obstructs the opening andsupports the flexible diaphragm around at least the periphery of theopening.

[0008] According to another embodiment of the present invention, amicrofluidic pump is provided including a fluid path, an inlet to thefluid path and an outlet to the fluid path. A first closing member and asecond closing member are each disposed along the fluid path between theinlet and the outlet, and a reservoir having a variable volume isdisposed along the fluid path between the first closing member and thesecond closing member. In this embodiment of the invention, the fluidpath has a cross-sectional dimension of less than about 500 μm.

[0009] According to another embodiment of the present invention, amicrofluidic system is provided including a flexible support, a flexiblematerial connected to the flexible support, and a fluid path within theflexible material having a cross-sectional dimension of less than about500 μm.

[0010] According to a further embodiment of the present invention, amethod for making a microfluidic system is provided. The method includesproviding a master corresponding to the microfluidic system, forming themicrofluidic system on the master, connecting a support to themicrofluidic system and removing the microfluidic system from themaster.

[0011] According to another embodiment of the present invention, amethod for opening a microfluidic valve is provided. The method includesproviding a microfluidic valve and a flow of a fluid through a fluidpath. The microfluidic valve includes the fluid path, an inlet and anoutlet to the fluid path, and a first closing member disposed along thefluid path between the inlet and the outlet. The method further includesdeflecting the closing member with the flow from a closed position to anopen position without the closing member sliding against any portion ofthe microfluidic valve. In this embodiment of the invention, the fluidpath has a cross-sectional dimension of less than about 500 μm.

[0012] According to another embodiment of the present invention, amethod for manipulating a flow of a fluid in a microfluidic system isprovided. The method includes providing a fluid path having across-sectional dimension of less that about 500 μm, initiating the flowof the fluid through the fluid path in a first direction, and inhibitingthe flow of the fluid through the fluid path in a second direction.

[0013] According to another embodiment of the present invention, amicrofluidic system includes a first fluid path, a second fluid path,and a first closing member comprised of a voltage degradable materialand disposed between the first and second fluid paths. In thisembodiment, one of the first and second fluid paths has across-sectional dimension of less than about 500 μm.

[0014] According to another embodiment of the present invention, amicrofluidic system includes a first fluid path, a second fluid path,and a first closing member comprised of a voltage degradable materialand disposed between the first and second fluid paths. In thisembodiment, the first closing member has a thickness of less than about50 μm.

[0015] According to another embodiment of the present invention, amicrofluidic device includes a substantially sealed fluid reservoir, afluid positioned within the fluid reservoir, a fluid path separated fromthe fluid reservoir by a closing member, a first electrode connected tothe fluid reservoir, and a second electrode connected to the fluid path.

[0016] According to another embodiment of the present invention, amethod of manipulating fluid flow in a fluidic system includes creatinga voltage difference between a first fluid path and a second fluid pathseparated by a closing member, the voltage being sufficient to form anopening in the closing member. The method further includes allowing afluid to flow between the first and second fluid paths.

[0017] According to another embodiment of the present invention, amethod of testing includes introducing a test fluid into a testreservoir. The method also includes creating a voltage differencebetween the test reservoir and a reagent reservoir containing a reagentand separated from the test reservoir by a closing member, the voltagedifference being sufficient to make an opening in the closing member.The method further includes allowing at least one of the test fluid andthe reagent to flow between the test reservoir and the reagentreservoir.

[0018] According to another embodiment of the present invention, amethod of making an opening in a fluidic system includes creating avoltage difference between a first fluid path and a second fluid pathseparated from the first fluid path by a closing member sufficient tomake an opening in the closing member.

[0019] Other advantages, novel features, and objects of the inventionwill become apparent from the following detailed description of theinvention when considered in conjunction with the accompanying drawings,some of which are schematic and which are not intended to be drawn toscale. In the figures, each identical or nearly identical component thatis illustrated in various figures is represented by a single numeral.For purposes of clarity, not every component is labeled in every figure,nor is every component of each embodiment of the invention shown whereillustration is not necessary to allow those of ordinary skill in theart to understand the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is an exploded, perspective view of one embodiment of amicrofluidic system according to the present invention, configured as avalve;

[0021]FIG. 2 is a cross sectional view through section line 2-2 of themicrofluidic system of FIG. 1, including a flow indicator;

[0022]FIG. 3 is a transparent, plan view of the microfluidic system ofFIG. 1;

[0023]FIG. 4 is an exploded, perspective view of one embodiment of amicrofluidic system according to the present invention, configured as avalve;

[0024]FIG. 5 is a cross sectional view through section line 5-5 of themicrofluidic system of FIG. 4, including flow indicators;

[0025]FIG. 6 is a transparent, plan view of the microfluidic system ofFIG. 4;

[0026]FIG. 7 is a photocopy of a photomicrograph of a lymph valve;

[0027]FIG. 8 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system according to the present invention, configured as avalve and including a flow indicator;

[0028]FIG. 9 is a photocopy of a photomicrograph of the microfluidicsystem of FIG. 8, including a flow indicator;

[0029]FIG. 10 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system according to the present invention, configured as avalve, including a flow indicator;

[0030]FIG. 11 is a photocopy of a photomicrograph of the microfluidicsystem of FIG. 10, including a flow indicator;

[0031]FIG. 12 is a transparent, plan view of one embodiment of amicrofluidic system of the present invention, configured as a pump;

[0032]FIG. 13 is a transparent, plan view of one embodiment of amicrofluidic system according to the present invention, including a flowindicator and cross-hatching to show the presence of a fluid within themicrofluidic system;

[0033]FIG. 14 is a transparent, plan view of the microfluidic systemillustrated in FIG. 13, also having a flow indicator and the presence offluid marked by cross-hatching;

[0034]FIG. 15 is a plan view of one embodiment of a microfluidic systemaccording to the present invention, configured as a pump, and includinga magnification of one portion of the microfluidic system;

[0035]FIG. 16 is a perspective, plan view of one embodiment of amicrofluidic system according to the present invention, including a flowindicator;

[0036]FIG. 17 is a perspective, plan view of the microfluidic systemillustrated in FIG. 16, including flow indicators;

[0037]FIG. 18 is a schematic view of one embodiment of a method formaking microfluidic systems of the present invention;

[0038]FIG. 19 is a perspective view of one embodiment of a microfluidicsystem according to the present invention;

[0039]FIG. 20 is a photocopy of a photomicrograph of a microfluidicsystem according to the present invention;

[0040]FIG. 21 is a graph of fluid resistance versus flow rate;

[0041]FIG. 22 is a graph of pressure drop versus flow rate;

[0042]FIG. 23 is plan view of one embodiment of a microfluidic systemaccording to the present invention, configured as a one-use valve;

[0043]FIG. 24 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system according to the present invention, configured as aone-use valve;

[0044]FIG. 25 is a photocopy of a photomicrograph of the microfluidicsystem of FIG. 24 in an open position;

[0045]FIG. 26 is a plan view of an embodiment a microfluidic system ofthe present invention, incorporating a plurality of one-use valves;

[0046]FIG. 27 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system of the present invention configured as a one-usevalve in an open position;

[0047]FIG. 28 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system of the present invention configured as a one-usevalve in an open position;

[0048]FIG. 29 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system of the present invention configured as a one-usevalve in an open position;

[0049]FIG. 30 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system of the present invention configured as a one-usevalve in an open position;

[0050]FIG. 31 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system of the present invention configured as a one-usevalve in an open position;

[0051]FIG. 32 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system of the present invention configured as a one-usevalve in an open position;

[0052]FIG. 33 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system of the present invention configured as a one-usevalve in an open position;

[0053]FIG. 34 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system of the present invention configured as a one-usevalve in an open position;

[0054]FIG. 35 is a photocopy of a photomicrograph of another aspect ofthe microfluidic system of FIG. 34;

[0055]FIG. 36 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system of the present invention configured as a one-usevalve in an open position;

[0056]FIG. 37 is a photocopy of a photomicrograph of another aspect ofthe microfluidic system of FIG. 36;

[0057]FIG. 35 is a top, plan view of one embodiment of a microfluidicsystem according to the present invention;

[0058]FIG. 39 is a photocopy of a photomicrograph of one embodiment of amicrofluidic system of the present invention; and

[0059]FIG. 40 is a photocopy of a photomicrograph of another aspect ofthe microfluidic system of FIG. 39.

DETAILED DESCRIPTION

[0060] The present invention is directed to a microfluidic system.“Microfluidic system,” as used herein, refers to a device, apparatus orsystem including at least one fluid path having a cross-sectionaldimension of less than 1 millimeter (mm). “Fluid path,” as used herein,refers to any channel, tube, pipe or pathway through which a fluid, suchas a liquid or a gas, may pass. “Cross-sectional dimension,” as usedherein, refers to the shortest distance that may be measured between anytwo opposed sides of a fluid path. However, in certain preferredembodiments, the longest distance that may be measured between any twoopposed sides of a fluid path is also less than the maximumcross-section for that embodiment.

[0061] In one embodiment, the microfluidic system includes a fluid path,an inlet to the fluid path, an outlet to the fluid path and a firstclosing member disposed along the fluid path between the inlet and theoutlet. As used herein, “closing member” refers to any structurespecifically adapted to selectively inhibit or prevent the flow of fluidthrough a fluid path or between fluid paths, reservoirs, and the like.Such a closing member has an open position and a closed position and maymove between these positions (either from open to closed or closed toopen) at least once. This definition specifically excludes structures,such a relatively thin wall between fluid paths, that are not intendedto have open and closed positions, but that may be opened or closedunder some circumstances, such as the application of a relatively highpressure. The microfluidic system according to this embodiment may beconstructed to function as a valve.

[0062] Referring now to the figures, and, in particular, to FIGS. 1-3,an embodiment of a microfluidic system that may be constructed to besuitable for use as a valve 12 will be described. This embodiment of amicrofluidic system 10 may include a fluid path 20, an inlet 22 to fluidpath 20, an outlet 24 to fluid path 20 and a closing member 30 disposedalong fluid path 20 between inlet 22 and outlet 24. Typically, inoperation of valve 12, a fluid is introduced, for example by pumping,into fluid path 20 through inlet 22. Following introduction, the fluidflows through fluid path 20 in a first direction toward outlet 24,passing closing member 30. However, if fluid flow changes direction, forexample due to a cessation of pumping, fluid is inhibited from flowingthrough fluid path 20 from outlet 24 to inlet 22 due to the action ofclosing member 30, which at least partially blocks its path. The actionof closing member 30 may be better understood with reference to theconstruction of valve 12.

[0063] Fluid path 20 may be constructed in any manner and of anymaterials that allow a fluid to flow through fluid path 20 withoutadversely affecting or being affected by the fluid. For example, fluidpath 20 may have any configuration or cross-sectional dimension thatallows passage of a fluid or fluids to be used with microfluidic system19 at an acceptable pressure drop. Preferably, the cross-sectionaldimension is as small as possible without inhibiting the flow of thefluid or fluids to be used with microfluidic system 10. For example,fluid path 20 may have a cross-sectional dimension of less than 1 mm,preferably less than 500 μm, more preferably less than 300 μm, stillmore preferably less than 100 μm and, most preferably, less that 50 μm.However, it should be recognized that the preferred cross-sectiondimension of fluid path 20 will vary with the fluid or fluids. Forexample, fluids, such as blood, including cells therein may sufferdamage to the cells if the cross-sectional dimension is too small. As afurther example, fluids having a relatively high viscosity may requireexcessive pumping pressure if the cross-sectional dimension is small.

[0064] The preferred configuration of fluid path 20 may vary withmicrofluidic system 10 and fluid or fluids to be used therein.Generally, fluid path 20 is preferred to be as straight and direct aspossible to minimize pressure drop and reduce damage to time sensitiveor shear sensitive liquids. However, in some instances, fluid path 20may be preferred to be longer or more convoluted than necessary, such aswhere fluid path 20 serves as a reactor or mixer wherein a residencetime is desired. Fluid path 20 may have any cross-section suitable foruse with the desired fluid or fluids. For example, the cross-section offluid path 10 may be polygonal, ovoid or of odd or irregular shape.

[0065] Fluid path 20 includes inlet 22 and outlet 24. Inlet 22 may beconstructed in any manner that allows fluid to be introduced into fluidpath 20. For example, inlet 22 may be a port, slit, funnel or otheropening. Inlet 22 may be adapted to mate with an additional fluid path20, pump or other device to facilitate the introduction of fluid intofluid path 20. Similarly, outlet 24 may be constructed in any mannerthat allows fluid to exit fluid path 20. For example, outlet 24 may be aport, slit or other opening. Outlet 24 may also be adapted to mate withan additional fluid path 20, pump or other device to facilitate passageof fluid from microfluidic system 10 into the additional fluid path 20,pump or other device.

[0066] Fluid path 20 may be constructed of any material or materialsthat will not adversely affect or be affected by fluid flowing throughfluid path 20. For example, fluid path 20 may be constructed of amaterial that is chemically inert in the presence of fluids to be usedwithin fluid path 20. Preferably, fluid path 20 is constructed of asingle material that is cheap, durable and easy to work with,facilitating field use and cost effective disposability. For example,fluid path 20 may be constructed of a polymeric material. Where fluidpath 20 is constructed of a polymer, the polymer may be selected based,for example, on its compatibility with the fluids to be used, itsdurability and shelf life, its cost and its ease of use. Preferably,fluid path 20 is constructed from poly(dimethlsiloxane) (“PDMS”). PDMSis a relatively inexpensive, durable, elastomeric polymer. Because PDMSis stable, fluid path 20 and other portions of microfluidic systemsconstructed of PDMS may have a shelf life of 6 months or more. PDMS isalso relatively easy to work with. It should be understood that whilepolymeric materials, and particularly PDMS, are preferred for theconstruction of fluid path 20, other materials, including conventionalsilicon chip materials, may be used to construct some or all portions offluid path 20. Other suitable materials include polymers described assuitable for use in fabricating a stamp in U.S. Pat. No. 5,512,131,which is hereby incorporated herein by reference in its entirety.

[0067] Closing member 30 may be constructed in any manner and of anymaterial or materials that allow it to selectively permit or inhibitfluid flow. One typical criteria for selecting whether to permit orinhibit fluid flow is fluid flow direction. For example, closing member30 may permit fluid flow in a first direction and inhibit fluid flow ina second direction as described previously. This type of closing member30 is referred to as a check valve. Where closing member 30 functions asa check valve, closing member 30 may be constructed in any manner suchthat it is opened by fluid flow in a first direction, and/or closed byfluid flow in a second direction. For example, closing member 30 may beconstructed such that it is pushed open by fluid flow in a firstdirection or pushed closed by fluid flow in a second direction.

[0068] Preferably, closing member 30 opens and closes without slidingagainst any portion of the microfluidic system as this may cause failureof closing member 30 due to mechanical damage or being caught in an openor closed position. Where closing member 30 is constructed such that itslides against any portion of the microfluidic system, some manner ofreducing the friction between closing member 30 and the portion of themicrofluidic system may be employed. For example, as illustrated inFIGS. 16, 17 and 19, the surfaces in contact with one another may benon-stick surfaces or may be treated with a suitable lubricant, such aspetroleum jelly (illustrated by the shaded regions of closing member30).

[0069] In some embodiments, such as those illustrated in FIGS. 1-9,closing member 30 is a flexible member. Where closing member 30 is aflexible member, closing member 30 may be constructed in any manner thatallows closing member 30 to be opened and closed as desired.

[0070] As illustrated in FIGS. 1-3, in some embodiments, closing member30 may be a flap. By flap it is meant a generally planar structureattached to a base, such that the structure may move relative to thebase. Where closing member 30 is a flap it may be constructed in anymanner that allows it to permit fluid to flow past closing member 30 ina first direction, but inhibits fluid flow in a second direction. Forexample, closing member 30 may be constructed such that it covers fluidpath 20 when closed. Where closing member 30 covers fluid path 20, fluidpath 20 may be constructed such that closing member 30 is allowed tomove in a first direction so that it does not cover fluid path 20 and isinhibited from moving in a second direction by the shape of fluid path20. For example, as illustrated in FIGS. 1-3, closing member 30 maycover a relatively narrow portion of fluid path 20, such as a seat 32,and reside in a relatively large portion of fluid path 20. Accordingly,as illustrated by flow indicator 50 in the lower portion of FIG. 2,fluid moving through fluid path 20 in a first direction may push closingmember 30 into the relatively large portion and into an open position.Conversely, as illustrated by flow indicator 50 in the upper portion ofFIG. 2, fluid moving through fluid path 20 in a second direction may notpush closing member 30 past seat 32 and, thus, cannot open it, rather,fluid pressure in the second direction acts to seal closing member 30 ina closed position, reducing the possibility of leakage.

[0071] Instead of being a single flap, closing member 30 may consist oftwo or more flaps. In some embodiments, such flaps, rather than closingagainst seat 32 of fluid path 20, may close against one another. Such aclosing member 30 a is illustrated in FIGS. 8, 9, 19 and 20. In FIG. 20fluid path 20 a and valve 12 a are open on their upper side for purposesof illustration. In the embodiment of FIGS. 8, 9, 19 and 20, valve 12 ais modeled after a lymphatic valve, such as that illustrated in FIG. 7.When fluid flows in a first direction, as indicated by flow indicator 50a in FIG. 8, the flaps of closing member 30 a are pushed apart, openingclosing member 30 a. Conversely, when fluid flows in a second direction,as indicated by flow indicator 50 a in FIG. 9, the flaps of closingmember 30 a are pushed against one another, closing and sealing closingmember 30.

[0072] In a valve having two closing members 30 a that close against oneanother, such as that illustrated in FIGS. 8, 9, 19 and 20, closingmembers 30 a may be attached to fluid path 20 a in any manner allowingthem to permit flow of fluid in a first direction and to inhibit flow offluid in a second direction. For example, closing members 30 a may beconnected to the sides, top and/or bottom of fluid path 20 a.Preferably, closing members 30 a have sufficient freedom of movement toeffectively come together to inhibit back flow, but not so much freedomthat they are easily bent over, twisted, or pushed aside by fluid flow.In one embodiment, closing members 30 a are connected to fluid path 20only at the sides of fluid path 20 a. Where specific versions of thisembodiment allow closing members 30 to twist or fall over with flow,another point of connection may be used, such as the top or bottom offluid path 20 a.

[0073] Where closing member 30 is a flap, it may be constructed in avariety of shapes. For example, closing member 30 may be rectangular, asillustrated in FIGS. 1-3, or may be constructed as another polygon, suchas a hexagon, a circle or a portion of a circle, such as a semicircle,or with an odd or irregular shape. Preferably, closing member 30 isroughly semicircular.

[0074] The manner and material of construction of closing member 30 maybe used to tailor the ease with which it is opened. For example, stifferclosing members 30, such as those that are thicker or constructed ofstiffer materials, will require more fluid pressure to open and mayprovide a better seal when closed, while more flexible closing members,such as those that are thinner or constructed of more flexiblematerials, may require less fluid pressure to open. It should also beappreciated that the degree of seal may also be dependant on the abilityof closing member 30 to conform to fluid path 20 when in a closedposition and, accordingly, if closing member 30 is too stiff to conformto fluid path 20, it may inhibit the seal rather than improving it.

[0075] As illustrated in FIGS. 4-6, in some embodiments, closing member30 b may be a diaphragm. By diaphragm it is meant a generally planarstructure attached at its edges to a base and having an opening therein.Where closing member 30 b is a diaphragm it may be constructed in anymanner that permits fluid to flow past closing member 30 b in a firstdirection, but not in a second. For example, closing member 30 b may beconstructed such that it covers fluid path 20 b but is allowed to movein a first direction, exposing an opening 34 b in closing member 30 b,through which fluid may flow. In this embodiment, closing member 30 b isinhibited from moving in a second direction to expose opening 34 b bythe shape of fluid path 20. For example, as illustrated in FIGS. 4-6,closing member 30 b may cover fluid path 20 b and be supported by a seat32 b that covers opening 34 b. Accordingly, as illustrated by flowindicators 50 b in the lower portion of FIG. 5, fluid moving throughfluid path 20b in a first direction pushes closing member 30 b away fromseat 32 b and into an open position. Conversely, as illustrated by flowindicators 50 b in the upper portion of FIG. 2, fluid moving throughfluid path 20 b in a second direction cannot push closing member 30 bpast seat 32 b and, thus, cannot open it. In fact, fluid pressure in thesecond direction acts to seal closing member 30 b in a closed position,pushing it against seat 32 b and reducing the possibility of leakage.

[0076] Where closing member 30 b is a diaphragm including opening 34,opening 34 b may be constructed in any manner that allows the passage offluid through opening 34 b. For example, opening 34 b may be constructedin a variety of shapes. For example, opening 34 b may be square orrectangular, as illustrated in FIGS. 4-6, or may be constructed asanother polygon, such as a hexagon, a circle or a portion of a circle,such as a semicircle, or with an odd or irregular shape. Seat 32 b maybe constructed in any manner that supports closing member 30 b around atleast the periphery of opening 34 b. For example, seat 32 b may also beconstructed in a variety of shapes. Preferably, the shape of seat 32 bcorresponds to the shape of opening 34 b to ensure adequate support ofclosing member 30 b. As is the case where closing member 30 is a flap,the manner and material of construction of closing member 30 b whereclosing member 30 b is a diaphragm may be used to tailor the ease withwhich closing member 30 is opened.

[0077] Closing member 30 c may also be a free-floating member. Whereclosing member 30 c is a free-floating member it may be constructed inany manner and of any material or materials that allow closing member 30c to selectively permit or inhibit fluid flow past closing member 30 c.For example, as illustrated in FIGS. 10 and 11, closing member 30 c maybe constructed such that it permits the flow of fluid in a firstdirection and inhibits the flow of fluid in a second direction. In theembodiment illustrated in FIGS. 10 and 11, closing member 30 c iscarried into and out of a narrow portion of fluid path 20 c, such asseat 32 c. When fluid is flowing in a first direction, as illustrated byflow indicator 50 c in FIG. 10, closing member 30 c is carried into anopen area, such that fluid can pass closing member 30 c. When fluidflows in a second direction, as illustrated by flow indicator 50 c inFIG. 11, closing member 30 c is carried by the fluid into seat 32 c offluid path 20 c, obstructing fluid flow.

[0078] Where closing member 30 c is a free-floating member, it mayconstructed to fit snugly within seat 32 c of fluid path 20 c,inhibiting leakage when fluid flow is in the second direction.Preferably, closing member 30 c is a spherical body and seat 32 c offluid path 20 is circular in cross-section; while other shapes andarrangements are possible, this arrangement ensures a snug fit betweenclosing member 30 c and fluid path 20 c, irrespective of the orientationof closing member 30 c.

[0079] Closing member may be constructed of any material or materialsthat allow it to selectively permit or inhibit fluid flow. Closingmember may also be made of a material that will not adversely affect orbe affected by a fluid or fluids in microfluidic system 10, such as amaterial that is inert with respect to the fluid or fluids for use inmicrofluidic system 10. The preferred material for closing member variesaccording to the nature of closing member. For example, where closingmember is a flexible member, such as flap or diaphragm, it is preferablyconstructed of a polymeric material, such as PDMS, as describedpreviously with respect to fluid path 20. Where closing member 30 c is afree-floating member, closing member 30 c is preferably constructed of amaterial that is easily moved by fluid flow and which may form a goodseal with the material of fluid path 20 c. For example, closing member30 c may be constructed of glass or other silicon-based material, apolymeric material, such as PDMS, or another relatively durable,lightweight material.

[0080] In another embodiment of the present invention, valve 12 d may beconstructed as a one-use valve. By “one-use valve” it is meant a valvethat, once opened, cannot be closed in the manner in which it wasopened. For example, in one embodiment of the invention, valve 12 d maybe a one-use valve opened by damage to closing member 30 d, such as avoltage difference across closing member 30 d that results in breakdownand consequent formation of an opening in closing member 30 d. In otherembodiments, modifying the temperature of closing member 30, or portionsthereof, may be used to open a one-use valve. Referring now to FIG. 23,one embodiment of valve 12 d configured as a one-use valve isillustrated. In the illustrated embodiment, a microfluidic systemincludes a first fluid path 20 d, a second fluid path 220 and a firstclosing member 30 d disposed between the first and second fluid paths 20d, 220.

[0081] First and second fluid paths 20 d, 220 may be arranged in anymanner that allows closing member 30 d to be formed between them. Forexample, first and second fluid paths 20 d, 220 may be arranged in a “T”shape, as illustrated in FIG. 23. As an alternate example, fluid paths20 d, 220 may be arranged end to end, effectively forming a single fluidpath broken by closing member 30 d. In some embodiments, such as thatillustrated in FIG. 26, multiple fluid paths may be separated from oneanother by multiple closing members 30 d. In the specific embodiment ofFIG. 26, ten fluid paths 20 d are separated from a single, central fluidpath 220 by ten closing members 30 d. The embodiment illustrated in FIG.23 may be part of a larger microfluidic system such as region 225 inFIG. 26. An arrangement such as that illustrated in FIG. 26 may beuseful for adding materials in a particular order, as required in someanalytical techniques.

[0082] Where valve 12 d is constructed as a one-use valve, closingmember 30d may be constructed to be in a closed position such that fluidflow past or through closing member 30 d is inhibited or eliminated. Forexample, closing member 30 d may include a substantially sealed barrier.Where closing member 30 d is constructed to inhibit or eliminate fluidflow, the dimensions of closing member may vary with the application andthe material from which closing member 30 is constructed. For example,closing member 30 d may be thick enough to remain closed until it isdesired to open it, but not so thick that it can not be openedconveniently, for example, that excessive voltage is required to do so.For example, for some materials, closing members 30 d between about 1and about 100 micrometers thick may be appropriate, for others, closingmembers 30 d between about 5 and about 50 micrometers thick may beappropriate, for others, closing members 30 d between about 15 and about40 micrometers thick may be appropriate, and for still others, closingmembers 30 about 20 micrometers thick may be desired.

[0083] While embodiments of closing member 30 d for use with a one-usevalve are illustrated herein as being generally planar and of uniformthickness, this disposition is not required, so long as closing member30 d may be opened as desired in a convenient way, e.g. using anacceptable voltage. For example, closing member 30 d may be shaped as ahemisphere, other regular shape, or an odd or irregular shape. In someembodiments, closing member 30d may be shaped such that it opens in adesired manner or produces an opening having a desired shape. Forexample, some portions of closing member 30 d may be thinner than otherportions of closing member 30 d, making formation of an opening morelikely in those portions.

[0084] Where closing member 30 d is constructed to inhibit or eliminatefluid flow, it may be constructed of any material or materials describedpreviously for formation of closing members as well as any othermaterial or materials that are compatible with the fluids to be used,substantially fluid tight, and capable of being opened by an acceptablevoltage. For example, closing member 30 d may be constructed of amaterial having a breakdown voltage of less than about 250 volts permicrometer, a breakdown voltage of less than about 150 volts permicrometer, a breakdown voltage of less than about 75 volts permicrometer, or a breakdown voltage of less than about 25 volts permicrometer. Materials with these characteristics can be readily selectedby those of ordinary skill in the art. For example, any polymer that isnot soluble in the liquid(s) to be used in the microfluidic system mayfind utility in particular embodiments. In a preferred embodiment,closing member 30 d is constructed from a material previously disclosedfor use in the formation of as fluid paths 20 d, 220. In anotherpreferred embodiment, closing member 30 d and fluid paths 20 d, 220 areformed in a single piece of material, such as PDMS (PDMS has a breakdownvoltage of 21 volts per micrometer). A material capable of being brokendown by application of an acceptable voltage is referred to herein as a“voltage degradable material.” As described herein, an acceptablevoltage may vary with the fluids to be used and other parameters of thespecific microfluidic system. For example, where bubble formation may beundesired, an acceptable voltage would inhibit bubble formation.

[0085] Referring now to FIGS. 23-25, a one way valve may be opened byproviding a voltage to closing member 30 d such that an opening 230 isformed therein. For example, a voltage greater than the breakdownvoltage of closing member 30 d may be provided such that opening 230 isformed in closing member 30 d. Voltage applied to closing member 30 dmay be of either negative or positive polarity. Voltage may be appliedin any manner so long as the voltage is directed to closing member 30 d.For example, an electrical source may be used to provide the desiredvoltage. The electrical source may be any source of electricity capableof generating the desired voltage. For example, the electrical sourcemay be a pizoelectrical source, a battery, or a device powered byhousehold current. In one embodiment, a pizoelectrical discharge from agas igniter was found to be sufficient to produce the desired voltage.

[0086] Voltage may be supplied to closing member 30 d through the use ofelectrodes 200. For example, electrodes 200 may be used to connectclosing member 30 d directly or indirectly to an electrical sourceand/or to a ground 210. In one embodiment, electrodes 200 may be placeddirectly in contact with closing member 30 d. In another embodiment,electrodes 200 may be placed in contact with fluid in fluid paths 20 d,220, so long as the fluid is sufficiently conductive to provide voltageto closing member 30d. Where indirect supply of voltage to closingmember 30 d is desired, the use of liquid fluids having sufficient ionicstrength to be conductive is generally preferred, though any conductivefluid may be used. Fluids having an ionic strength of 10 mM (millimolar)and 166 mM were found to be suitable for transmitting the voltage, withthe higher ionic strength fluid generating a larger opening.

[0087] Electrodes 200 may be constructed in any manner that allowselectricity to be transmitted directly or indirectly to closing member30 d. For example, electrodes 200 may include any conductive materialcompatible with the fluids and materials to be used in the microfluidicsystem. Higher conductivity materials that transmit voltage quickly toclosing member 30 d, resulting in shorter voltage ramp times, and largeropenings, as will be described below, are generally preferred. Examplesof materials that may be form suitable electrodes 200 include conductivemetals such as steel, platinum, silver, gold and copper as well as otherconductive materials, such a conductive polymeric materials.

[0088] In microfluidic systems having more than one closing member thatmay be opened, the fluid path on the opposite side of the closing memberto be opened, and to which voltage is to be applied, may be grounded.For example, in microfluidic systems including multiple closing membersconnected to a single fluid path 220, such as illustrated in FIG. 26,where voltage is to be applied to first fluid path 20 d, second fluidpath 220 may be grounded to inhibit opening of more than one closingmember 30 d where this opening of more than one closing member is notdesired. In a similarly configured embodiment where voltage is appliedto second fluid path 220 rather than first fluid path 20 d, first fluidpath 20 d may be grounded to promote opening of first fluid path 20 dand not other fluid paths connected by closing members to second fluidpath 220.

[0089] The voltage to open a particular closing member may varydepending on the construction of the closing member and itssurroundings. For example, the thickness of the closing member and itsbreakdown voltage may affect the opening voltage. The ionic strength offluid in fluid paths 20 d, 220 may also affect the opening voltage. Thetheoretical minimum opening voltage required if delivered directly tothe closing member is the product of the break down voltage of thematerial(s) forming the closing member and the thickness of the closingmember. For, example, for PDMS closing members 20 micrometers thick andhaving a breakdown voltage of 21 volts per micrometer, the theoreticalminimum pulse of voltage is 20 micrometers times 21 volts per micrometeror 420 V.

[0090] Actual voltages may be applied to closing member 30 d that aregreater than the theoretical minimum opening voltage. Applying voltagesgreater than the theoretical minimum may decrease the length of thevoltage pulse required to create opening 230. For example, for 20micrometer thick PDMS closing members, openings 230 were complete inless than 1 second for voltages above 1 kV, while a voltage of 2 kVcreated opening 230 in 50 milliseconds and a voltage of 5 kV createdopening 230 in 20 milliseconds. The actual voltage at breakdown ofclosing member 30 d may be less than the applied voltage becausebreakdown and the creation of opening 230 may occur during the rampingperiod. For example, the above-described 2 kV and 5 kV pulses actuallyresulted in breakdown at 1.8 kV and 3.4 kV, respectively, because thebreakdown occurred during the ramping period.

[0091] The length of a voltage pulse may be any amount of time thatproduces the desired opening 230 in closing member 30 d. For somematerials and conditions, the size of opening 230 may be independent ofthe length of the voltage pulse, so long as a minimum pulse length isused. For example, 50 millisecond, 60 millisecond and 1 second pulses of2 kV across a PDMS closing member were found to produce openings 230 ofapproximately the same size, as illustrated in FIGS. 27-29.

[0092] Opening 230 may take any form that allows the desired amount offluid to pass between fluid paths 20 d, 220. For example, opening 230may be sized to allow the desired amount of fluid to pass between fluidpaths 20 d, 220. For example, where a lower flow rate is preferred,opening 230 may be relatively small, while, where a higher flow rate ispreferred, opening 230 may be larger. Depending on the embodiment, holesfrom about 2 micrometers in average diameter to about 50 micrometers inaverage diameter may be useful, and even smaller, or larger, openings230 may be desired in particular embodiments. The size of opening 230may be limited only by the size of closing member 30 d, and may evenexceed thee size of closing member 30 in some embodiments.

[0093] The voltage, voltage ramp rate and ionic strength of fluid withinfluid paths 20 d, 220 may affect the size of opening 230. Specifically,higher voltages, faster ramp rates and higher ionic strength fluidstypically result in larger openings. For example, constant voltage ramprates, such as 500 V/50 ms, resulted in smaller openings than those whenthe ramp rate increased exponentially with time. In one particularembodiment where the voltage increased exponentially with time, thevoltage as a function of time was roughly:

V(t)=5000*(1−exp(−t/τ)

[0094] where t is time, V is voltage and r is resistance timescapacitance (RC), which was 16 ms. Similarly, rate of distribution ofcharge from the electrode to the closing member is dependant on theresistivity of any fluid therebetween; increasing the ionic strength(decreasing the resistivity) of the fluid thus effectively shortens theramp rate of the electrical source, resulting in a larger opening.Accordingly, a desired size of opening 230 may be provided by adjustmentof these values.

[0095] Opening 230 may be any shape that allows the desired amount offluid to pass between fluid paths 20, 220 and need not be a single hole.For example, opening 230 may be of odd or irregular shape or may be of aparticular shape dictated by the shape of closing member 30 d. Asanother example, opening 230 may consist of a plurality of smaller holesin closing member 30 d. With particular conditions and materials, suchas PDMS, opening 230 may generally be formed as a series of fissures orholes. Without wishing to be limited to any particular theory, it isbelieved that these fissures are caused by stresses occurring duringbreakdown, such as gas evolution and expansion, thermal stress, andchemical bond breaking. Suitable combinations of voltage and voltagepulse length to achieve an opening of a desired size/shape may bedetermined for a particular arrangement using routine experimentation.

[0096] Where a voltage pulse is applied to a closing member to open it,bubbles of gas may be formed in liquid-containing fluid paths adjacentthe closing member after the connection is opened. The magnitude andlength of a voltage pulse may affect the size of these bubbles. Lowermagnitude, shorter voltage pulses may reduce bubble formation, whilelarger magnitude, longer voltage pulses may promote bubble formation.Without wishing to be limited to any particular theory, it is believedthat high local temperatures accompanying the electrical breakdown ofthe closing member results in bubbles. Where bubbles are formed, suchbubbles may dissipate. For example, bubbles formed from breakdown of 20micrometer thick PDMS closing members may dissipate after about 30seconds. Bubbles may also be moved out of the fluid paths, for exampleby pumping via applied pressure. Such pumping may be performed manuallywhere the microfluidic system includes flexible fluid paths.

[0097] Microfluidic systems incorporating one-use valves may be used inany application where it is desired to keep the fluid paths separateduntil a particular time. For example, fluids, such as reagents,analytes, and buffers may be stored in fluid paths. In microfluidicsystems having multiple channels separated by one-use valves, openingthe valves in a predetermined sequence may allow complex analyses andchemical reactions to be performed. For example, a microfluidic devicefor use in ELISA (Enzyme-Linked Immunosorbent Assay) experiments, whichrequire several solutions be added separately and in a specific order,may be created using the one-use valves of the present invention; thesesolutions may include capture antibody, blocking agent, sample,antibody-enzyme conjugate, and enzyme substrate. Microfluidic fluidpaths are especially suitable for ELISA because the relatively highratio of surface area to volume reduces the effect of mass transportlimitations. Furthermore, where PDMS is used to create some or all ofthe microfluidic system, some common capture antibodies for use withELISA are readily adsorbed onto oxidized PDMS. Other potential uses formicrofluidic systems incorporating one-use valves include chemicalanalysis, drug delivery, and chemical synthesis.

[0098] Microfluidic system 10 as thus far described is suitable for useas a valve. Referring now to FIGS. 12-15, microfluidic system 10 e maybe expanded for use as a pump. In one embodiment of microfluidic system10 e for use as a pump, microfluidic system 10 e further includes asecond closing member 31 disposed along fluid path 20 e between inlet 22e and outlet 24 e and a reservoir 40 disposed along fluid path 20 ebetween first closing member 30 e and second closing member 31. As usedherein, “reservoir” refers to any structure generally intended to hold avolume of fluid. Reservoirs may also function as fluid paths in thatfluid may pass through them at times and fluid paths may sometimesfunction as reservoirs in that they may sometimes be used to hold fluid.Second closing member 31 may be constructed in any manner and of anymaterial or materials previously described for closing member 30 e.According to the embodiments of the invention illustrated in FIGS.12-15, closing members 30 e, 31 operate as check valves 12 e. In thisembodiment, check valves 12 e allow fluid to flow through fluid path 20e only in the first direction, from inlet 22 e to outlet 24 e.

[0099] Reservoir 40 may be constructed in any manner and of any materialor materials that do not adversely affect, and are not adverselyaffected by, fluid in reservoir 40 and allow the pressure in reservoir40 to be varied. Increasing the pressure in reservoir 40 beyond thepressure in fluid path 20 e may force fluid within reservoir 40 out ofreservoir 40. However, check valves 12 e allow fluid to flow only in thefirst direction (illustrated by flow indicators 50 e in FIG. 12).Accordingly, fluid will flow toward outlet 24 e, but will not bepermitted to flow toward inlet 22 e. Conversely, decreasing the pressurewithin reservoir 40 to below the pressure within fluid path 20 e maydraw fluid into reservoir 40. Once again, check valves 12 e allow fluidto flow only in the first direction. Accordingly, fluid will be drawnfrom the direction of inlet 22 e into reservoir 40, but will not bepermitted to flow in the second direction from outlet 24 e towardreservoir 40. It will now be apparent that by alternately increasing anddecreasing the pressure in reservoir 40 it is possible to pump fluidthrough fluid path 20 e from inlet 22 to outlet 24 e. The speed of thepressure changes and the volume of reservoir 40 are generally directlyproportional to the speed the fluid is pumped.

[0100] In a preferred embodiment, reservoir 40 is constructed such thatthe volume of reservoir 40 is variable, allowing the pressure withinreservoir 40 to be varied by varying the volume of reservoir 40. Wherethe volume of reservoir 40 is variable, it may be variable due to theflexibility of reservoir 40. Accordingly, pressure applied externally toreservoir 40 may deflect reservoir 40, decreasing its volume andcorrespondingly increasing the pressure within it and pumping fluidtoward outlet 24 e. If reservoir 40 is elastomeric, when the externalpressure is removed from reservoir 40 it will return to its originalshape, increasing its volume, decreasing the pressure within it, anddrawing fluid from inlet 22 e. Accordingly, reservoir 40 is preferablyconstructed from an elastomeric material, such as the polymers discussedpreviously. Reservoir 40 may be constructed according to the rapidprototyping technique described later herein. Where desired, thisprocess may be modified to increase the volume of reservoir 40. Forexample, the portion of the master corresponding to the reservoir may beconstructed to be thicker than the rest of the master, leading to alarger reservoir 40. In one embodiment, the portion of the mastercorresponding to the reservoir may have an additional material, such asan epoxy, added thereto to increase its thickness and, thus, the volumeof reservoir 40. Such an arrangement may also allow the upper wall ofreservoir 40 to be made thinner, allowing its volume to be more easilyadjusted.

[0101] As illustrated in FIGS. 12 and 15, microfluidic system 10 e foruse as a pump may include more than one check valve 12 e along flow path20 e before and after reservoir 40. For example, two, three or morecheck valves 12 e may be included on either side of reservoir 40 toinhibit back-flow during actuation of the pump. In one embodiment,similar to that illustrated in FIG. 15, a pressure of about 150 Pa(Pascals) was generated in the adjacent fluid paths by compression ofreservoir 40 with a flow rate of about 0.66 microliters per stroke.

[0102] Where microfluidic system 10 e for use as a pump includesreservoir 40 having a variable volume, the volume may be varied in anymanner that produces the desired pumping function. For example, wherereservoir 40 is constructed of an elastomeric material, the volume ofreservoir 40 may be varied manually. Such a reservoir may be sized andshaped to complement a thumb or finger; for example, it may be roughlyoval and about 2 square cm. In another example, microfluidic system 10 emay be constructed with a second reservoir adjacent to reservoir 40 anda fluid, such as air, may be pumped into and out of the second reservoirsuch that it compresses or expands the second reservoir andcorrespondingly expands or compresses reservoir 40 due to its proximityin the elastomeric material.

[0103] Microfluidic system 10 according to the present invention may beflexible. For example, where components of microfluidic system 10 areconstructed of a flexible material some, or all, of microfluidic system10 may be flexible. Some embodiments of microfluidic system 10 of thepresent invention may be constructed entirely of elastomeric polymers,such as PDMS. Flexible microfluidic systems according to the presentinvention are resistant to breakage and may be bent or twisted. Forexample, microfluidic system 10 may be constructed for use as anassaying device for fieldwork, such as on-site environmental testing ormedical diagnosis. In such a device a lengthy fluid path 20 may bedesirable to promote a reaction or separation, but may interfere withportability. According to the present invention the microfluidic system10 could be coiled, allowing a lengthy fluid path 20 to be containedwithin a relatively small space.

[0104] In some instances where microfluidic system 10 is flexible, itmay be desirable to increase its structural stability and ability toresist damage or deformation. In this case, it is preferred to providesupport to microfluidic system 10. Preferably, support is providedwithout compromising the flexibility of microfluidic system 10. Forexample, microfluidic system 10 may be supported on a support 110 (seeFIG. 18). Support 110 may be flexible and may be constructed in anymanner and of any material or materials that provide the desired degreeof support, stability and flexibility to microfluidic system 10. Forexample, support 110 may be constructed of a polymer.

[0105] Support 110 may be connected to microfluidic system 10 in anymanner and using any materials that provide the desired connection. Forexample, in some embodiments, it may be desired to form a reversibleconnection to microfluidic system 10, and, in others, an irreversibleconnection. Support 110 may be connected to microfluidic system 10 usingan adhesive, such as a conventional pressure-sensitive adhesive. In someinstances, conventional adhesive tapes may perform as suitable supports.For example, an adhesive tape with a silicone adhesive on a polyesterbacking is a suitable support 110 for some applications.

[0106] Microfluidic system 10 according to the present invention may beconstructed using any method that will repeatably produce microfluidicsystem 10 having the desired structure and functionality. For example,microfluidic system 10, or portions of microfluidic system 10, may beconstructed by conventional etching techniques known in the art.Preferably, microfluidic system 10 is constructed according to themethod of the invention, as disclosed herein.

[0107] Referring now to FIG. 18, in one embodiment, a method for makinga microfluidic system includes providing a master 100 corresponding tomicrofluidic system 10, forming microfluidic system 10 on master 100,connecting support 110 to microfluidic system 10, and removingmicrofluidic system 10 from master 100.

[0108] The act of providing master 100 corresponding to microfluidicsystem 10 may be performed in any manner that produces master 100corresponding to microfluidic system 10. For example, master 100 may beproduced by conventional etching techniques. More specifically, in oneembodiment, master 100 corresponding to microfluidic system 10 may beconstructed by producing high resolution transparencies according tocomputer designs, such as CAD drawings, corresponding to the design ofmicrofluidic system 10. These transparencies may then be used as maskswhen transferring a pattern into negative photoresist by conventionalphotolithography, yielding a master with positive relief of fluid paths20 and other features of microfluidic system 10. This method isdescribed in more detail in “Rapid Prototyping of Microfluidic Systemsin Poly(dimethylsiloxane).” Anal. Chem. 1988, 70, 4974-4984., which ishereby incorporated by reference in its entirety. In FIG. 18, twomasters 100 are illustrated, one corresponding to a flow path 20 for usewith diaphragm closing member 30 and the other corresponding todiaphragm closing member 30.

[0109] The act of forming microfluidic system 10 on master 100 may beperformed in any manner that produces microfluidic system 10 capable ofproviding desired fluid flow properties. For example, microfluidicsystem 10 may be cast or molded onto master 100. In one embodiment, amoldable polymer or prepolymer may be placed in contact with master 100and polymerized or cured such that it has sufficient rigidity to providedesired fluid flow properties in microfluidic system 10. The desiredstiffness may vary with the intended application for microfluidic system10. For example, if microfluidic system 10 is desired to be flexible, itis preferred to use a flexible polymer. In a preferred embodiment,forming microfluidic system 10 on master 100 is performed by replicamolding against the master and the preferred material is PDMS.

[0110] As illustrated in FIG. 18, in some embodiments, a secondarymaster 120 may be used. For example, where microfluidic system 10includes flow path 20 with features on both sides of microfluidic system10, such as flow path 20 or opening 34 passing through microfluidicsystem 10, a secondary master 120 may be used. Accordingly, secondarymaster 120 may corresponding to the side of microfluidic system 10facing away from master 100. In some embodiments, such as thatillustrated in FIG. 18, secondary master 120 may include a flat sheet tosqueeze excess material out of master 100 and to ensure that flow path20 passing through microfluidic system 10 is not blocked by suchmaterial. Pressure may be exerted upon secondary master 120 to ensurethe desired amount of material is squeezed out of master 100. Thepressure exerted may depend, for example, on the type of material beingused to form microfluidic system 10 and the amount of material that mustbe squeezed out of master 100. In some instances, pressure of about 1pound per square inch (psi) may be applied to secondary master 120.Preferably, secondary master 120 is formed of a material that is easilyremoved from microfluidic system 10, such as Teflon®polytetrafluoroethylene (“PTFE”) available from DuPont Corporation ofDelaware. Secondary master 120 may be treated to ensure that it issmooth.

[0111] The act of connecting support 110 to microfluidic system 10 maybe performed in any manner that provides the desired degree ofconnection. For example, in some embodiments, it may be desired to forma temporary connection only strong enough to pull microfluidic system 10from master 100, while in other embodiments it may be desired to for anirreversible connection as has been previously discussed.

[0112] The act of removing microfluidic system 10 from master 100 may beperformed in any manner that will not damage microfluidic system 10. Forexample, support 1 10 may be lifted away from master 100, pullingmicrofluidic system 10 with it. After microfluidic system 10 is removedfrom master 100, support 110 may remain attached the microfluidic system10, serving as a substrate, or support 110 may be used to facilitatetransfer to another substrate or microfluidic system, as will bediscussed below. Where support 110 is used to facilitate transfer ofmicrofluidic system 10, as illustrated for closing member 30 in FIG. 18,support 110 may be removed from microfluidic system 10 when the transferhas been accomplished. The act of removing support 110 from microfluidicsystem may be performed in any manner that does not harm microfluidicsystem 10. For example, if an adhesive was used to connect support 110to microfluidic system 10, a material may be used to dissolve theadhesive without dissolving or damaging microfluidic system 10. Forexample, an appropriate solvent may be used.

[0113] Microfluidic system 10 formed by the method of the invention maybe combined with other microfluidic systems 10 to form largermicrofluidic system 10. For example, a more complex microfluidic system10, such as a microfluidic system intended for use as a valve or a pump,may be formed as layers according to the method of the invention andthen connected. In most embodiments, it is preferred that suchconnections between layers of microfluidic system 10 be substantiallyirreversible. For example, FIG. 18 illustrates making a portion ofmicrofluidic system 10 configured as a diaphragm valve, such as thatillustrated in FIGS. 4-6, from two microfluidic systems 10, oneincluding fluid path 20 and the other configured as closing member 30.

EXAMPLES Example 1

[0114] A rapid prototyping method was used for the design andfabrication of microfluidic valves and pumps. First, high-resolutiontransparencies were produced from a CAD file containing a design offluid paths. These transparencies were used as masks in transferring thedesign into negative photoresist by conventional photolithography,yielding a master with positive relief of fluid paths.

[0115] The valves were made from two molded PDMS bas-relief plates and amembrane. As illustrated in FIG. 18, the parts were fabricatedseparately and later assembled to complete the valves. First, one PDMSbas-relief plate was constructed (a) by replica molding against themaster using procedures known in the art.

[0116] A PDMS membrane was constructed (b) by casting and curing thePDMS prepolymer between a master and a secondary master in the form of aTeflon® PTFE sheet (1 mm thick Teflon® FEP, DuPont, Del). Modestpressure (1 psi) was applied to the secondary master/PDMS/mastersandwich while curing to squeeze out excess PDMS prepolymer. The PDMSmembranes were 25-100 μm thick, as thick as the negative resist (SU-8,MicroChem, MA) used in making the master. A master with photoresistposts, as shown in FIG. 18, was used to obtain PDMS membranes withthrough-holes. Prior to use as the secondary master, the Teflon(t PTFEsheet was molded against a flat Si wafer surface at 300° C. (T_(g)=270°C.) to obtain a smooth surface.

[0117] After curing, the secondary master was removed to leave behindthe PDMS membrane attached to the master. A pressure sensitive adhesive(PSA, Furon M803 adhesive tape with silicone adhesive on polyester back,Furon, Conn.) was applied on the PDMS membrane as a support. Due tostronger adhesion between the support and PDMS compared to that betweenPDMS and master, it was possible to transfer the membrane from themaster to the support by peeling the support away from the master. Themembrane, once transferred onto the support, could be handled withoutdistortion. The support was removed by applying appropriate solvents(acetone or ethanol) after manipulation.

[0118] The support/PDMS membrane was placed in an aligner and bonded (c)with PDMS bas-relief plate to form the lower part of the valve. Thealigner was constructed from a set of x-y-z micrometer stages mounted ona translation post. The patterned PDMS membranes (supported on supportor master) and bas-relief plates were placed on the top and bottommicrometer stages and aligned using a stereo microscope. Irreversiblebonding of the PDMS pieces was achieved by surface modification byoxygen plasma treatment. After alignment, the assembly containing thealigner and the PDMS pieces were placed in oxygen plasma (Harrick, Pa.)for 30 sec. (60 W, 200 mTorr). The PDMS pieces were brought into contactimmediately after they were removed from the plasma generator. Complete,functional valves were fabricated by repeating (c) with anotherbas-relief plate that forms the top fluid path.

Example 2

[0119] Microfluidic systems incorporating one-use valves wereconstructed of two layers, one layer with embedded fluid paths and oneflat layer. To fabricate the layer with the fluid paths, PDMS was moldedagainst a photolithographic master produced by rapid prototyping andcomprising a positive relief of photoresist on a silicon wafer. (See“Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane).”Anal. Chem. 1988, 70, 4974-4984.) Holes were cut in the PDMS usingcircular punches to form fluid reservoirs. The layer with fluid pathswas sealed against a flat piece of PDMS or a glass slide by oxidizingboth layers in an air plasma and then bringing them into conformalcontact. The plasma oxidation used in the sealing process rendered thechannels hydrophilic, and they were readily filled with aqueous buffer.Dead-end channels filled by capillary action in less than 5 minutes. Themicrofluidic systems included of at least two disconnected channelsseparated by 20μm closing members, for example as shown in FIGS. 24-25.This separation was limited by the resolution of the printing used inthe first step of rapid prototyping. A higher-resolution printer wouldallow thinner separations and lower voltages to operate. The channelswere 50 or 100 μm wide and 60 μm tall.

Example 3

[0120] The valves constructed in Example 1 were tested for long-termoperation using an external three-way electromechanical valve (LeeValve, Westbrook, Conn.) connected to a pressurized air source(functionality of the valves was tested with liquids before and afterlong-term testing with pressurized air). The inlet of the valve wasconnected to the electromechanical valve which pressurized and ventedthe valve at 10 Hz. The outlet of the valve was submerged in a waterbath and the generation of a bubble with each opening and closing wasmonitored. The PDMS diaphragm valves were tested continuously for 105openings and closings. The valves did not show any marked deteriorationand were fully functional when tested with fluids (water).

[0121] Both diaphragm and flap valves were still fully functional whentested after storing them for six months in the open laboratory. Onlythe valves that had dried solids around the membrane that could not beremoved were not functional. The ones with particle/deposit that werewashed and cleaned functioned without any problems.

Example 4

[0122] In order to demonstrate the effectiveness of one embodiment of avalve according to the present invention in reducing back flow, flowtests were performed on a valve as illustrated in FIGS. 8, 9, 19 and 20.Closing members 30 a were connected to three of four walls (the twosides and the bottom) of a rectangular fluid path 90 micrometers highand 150 micrometers wide. The width of the fluid path in the area ofclosing members 30 a was greater than the width elsewhere in the fluidpath. Closing members 30 a were designed not to touch at rest, with nofluid flowing, and were separated by 20 micrometers. Closing members 30a were the same height as fluid path 20 (90 micrometers) and taperedfrom the walls of fluid path 20 to a width at their ends ofapproximately 20 micrometers. The dimensions of the valves depicted inFIGS. 8-9 are about 40% larger than those used in this example. The topof each closing member 30a where it abutted the top of fluid path 20 wascoated with petroleum jelly for lubrication, as illustrated by shadingin FIG. 19.

[0123] For the particular dimensions of this embodiment, a back pressureof approximately 1.7 kPa (kilopascals) was required to bring togetherclosing members 30. Back flow rates between 0 microliters per second and0.3 microliters per second (0.3 microliters per second corresponded to1.7 kPa) resulted in slight opening of closing members 30 a resulting inreduced pressure drop. However, as soon as a back flow of 0.3microliters per second was reached, the closing members were pushedtogether by the pressure.

[0124]FIGS. 21 and 22 are graphs of the resistance of the valve versusthe flow rate through the valve and pressure drop across the valveversus the flow rate through the valve, respectively. FIG. 22illustrates that the partial closure of closing members 30 a is moreabrupt than their opening. Based on the slopes of pressure drop versusflow rate, three states of the valve may be defined: “closed” at largenegative (back flow) flow rates, “neutral” at low flow rates, and “fullyopen” at relatively large positive flow rates. The linear regression ofthe pressure drop versus flow rate curve is an alternative measure ofthe fluidic resistance over each of the regions. The resistance of thevalve was 20×10⁶, 4.4×10⁶, and 2.4×10⁶ μL-1 in the closed, neutral, andopen positions, respectively. The resistance to flow across the valve ofthis embodiment was therefore approximately 8 times greater for backflow than for forward flow. This demonstrates that this embodiment ofthe valve of the present invention is effective as a check valve.Without wishing to be limited to any particular theory, it is believedthat the inhibition, as opposed to nearly complete stoppage, of flow wasdue to the gap at the bottom of the fluid path where the closing membersare not free to move together, as well as gaps at the top of the fluidpath where the closing members are not connected.

Example 5

[0125] In order to demonstrate the effectiveness of the seal between thecomponents of the embodiment of the microfluidic system described inExample 4, the microfluidic system was exposed to increasing fluidpressures. The microfluidic system withstood fluid pressures from300-500 kPa, demonstrating that an effective seal was formed between thecomponents.

Example 6

[0126] In order to demonstrate the durability of the microfluidic systemdescribed in Example 4, the microfluidic system was set to open andclose through 40,000 cycles over 11 hours. The microfluidic systemsuffered no obvious fatigue or decrease in performance as a result,demonstrating the durability of the microfluidic system.

[0127] Also to demonstrate durability of the microfluidic systemdescribed in Example 4, the microfluidic system was filled with a sodiumchloride solution and the liquid was evaporated leaving a salt crust onthe fluid paths and closing members. Function was restored uponrehydration, again demonstrating durability of the microfluidic system.

Example 7

[0128] In order to determine the effect of applied voltage on openingsize and minimum pulse length for one-use valves, experiments wereperformed at several voltages. PDMS closing members 20 micrometers thickwere used in the experiments. After the openings were formed,fluorescein was pumped through the openings. The results of some ofthese experiments are shown in FIGS. 30-33, which are photocopies offluorescent micrographs of openings formed by voltages of 1 kV, 2 kV, 5kV and 10 kV, respectively. Voltages from 1 kV to 5 kV were applied by acommercial power supply (CZE1000R, Spellman High Voltage, Hauppauge,N.Y.) controlled by an analog output board (PCI-MIO-16XE-50, NationalInstruments, Austin, Tex.) and LabVIEW ® software (NationalInstruments), while the approximately 10 kV voltage was applied by a gasigniter (Weber-Stephen Products, Burlington, Canada).

[0129] Openings were formed in one second for 1 kV, 50 milliseconds for2 kV, 20 milliseconds for 5 kV, and 20 microseconds for 10 kV. Voltagesgreater than 2 kV achieved reproducible openings, but the size and shapeof the openings were variable. Where openings were formed, theapproximate average diameters openings were 5 micrometers for 1 kV, 20micrometers for 2 kV, 50 micrometers for 5 kV and 5 micrometers for 10kV (supplied by the gas igniter). Without wishing to be limited to aparticular theory, it is believed that the smaller hole at 10 kV was dueto the very short duration of the pulse generated by the igniter or tothe fact that that the power generated by the igniter was less than thatproduced by the commercial power supply. The pressure required to pumpthe fluorescein through the openings was greatest for the opening formedby 1 kV and least for the opening formed by 5 kV. The results of theseexperiments demonstrate that higher voltages generally produce largerholes and require shorter pulses to form openings.

Example 8

[0130] Experiments were performed in order to determine the effect ofthe ionic strength of a fluid positioned within fluid paths 20 d, 220,on the opening of a one-use valve. As ionic fluids PBS (10 mM(millimolar) phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.4, I (Ionicstrength) approximately 166 mM) and Tris-Gly (25 mM Tris—192 mM Gly, Iapproximately 10 mM) containing 0.1 micromolar fluorescein were used.The experiments were performed in a microfluidic system such as thatillustrated in FIG. 23, where two fluid paths 20 d, 200 are separated bya PDMS closing member 30 d approximately 20 micrometers in thickness. Avoltage of −5 kV was applied to fluid path 20 d, with fluid path 220grounded.

[0131] The results of these experiments are shown in FIGS. 34-37,wherein white regions indicate the presence of fluorescein, thus, fluid.FIGS. 34 and 36 show horizontal sections 2.6 micrometers thick of theclosing members (See FIG. 23). FIG. 34 shows the opening formed with aPBS buffer. The opening is halfway between the PDMS-glass seam (top thefluid path) and the bottom of the fluid path. FIG. 36 shows the middleof an opening formed with the Tris-Gly buffer (approximately 5micrometers from the PDMS bottom of the channel). FIGS. 35 and 37 showvertical sections (2 micrometers thick) of connections half way betweenfluid paths 20 d, 220 (10 micrometers from either channel). Closingmember 30 d is positioned between a glass support 500 and a PDMS base501. FIG. 35 shows an opening formed with PBS. This opening extendedfrom the top to the bottom of the fluid path and consisted primarily oftwo fissures, each about 10 micrometers in average diameter. FIG. 37shows the approximately 10 micrometer average diameter opening (heightof about 30 micrometers) formed with the Tris-Gly buffer. Theseexperiments demonstrate that a buffer with a higher ionic strengthproduces larger holes than a buffer with a lower ionic strength.

Example 9

[0132] In order to determine the minimum voltage required to form anopening, experiments were performed using a 20 micrometer thick PDMSclosing member with both Tris-Gly and PBS buffers (as used in Example8). The theoretical voltage to open such a closing member is 420 V.Current flow was observed at 500 V, but formation of openings was notreproducible at this voltage. Openings formed more frequently as thevoltage was increased to 600 V, 750 V, and 1 kV, but again the resultswere not reproducible. In some cases it was possible to open aconnection by repeatedly applying a given voltage or applying a highervoltage across a smaller opening. Without wishing to be limited to anyparticular theory, it is believed that these variations are due toheterogeneities in the PDMS. These experiments demonstrate that voltageshigher than the theoretical minimum may be required to produce openingsin some instances and that higher voltages are more likely to formopenings.

Example 10

[0133] In order to demonstrate that the one-use valves of the presentinvention can be used to produce relatively complex microfluidicsystems, an ELISA device was developed. This device is illustrated inFIG. 38 and included 5 reservoirs 401-405. A “T” shaped fluid path 20 dconnected reservoirs 403-405; each of these reservoirs had an averagediameter of 4 mm. Reservoir 405 served as a reservoir for waste, andreagents, sample, and rinse solution were added in reservoirs 403 and404. Reservoirs 401 and 402, which were approximately 1 mm in averagediameter, were disconnected from fluid path 20 d, but were connected tosyringes containing reagents and to closing member 30 d by fluid paths220. A detection chamber 406 in fluid path 20 d was used to present alarger surface (600×600 micrometers) area for observation. Coupling fromthe syringes (1 mL, Henke-Sass, Wolf, Tuttlingen, Germany) to the devicewas accomplished by using polyethylene tubing (outer diameter 1.09 mm,inner diameter 0.38 mm, Becton Dickinson, Franklin Lakes, N.J.) that waspressure fit into reservoirs 401, 402. Flow of the coating antibody,rinsing solution, and sample was by hydrodynamic pressure from 50microliters of fluid in reservoirs 403 and 404 to reservoir 405.

[0134] Antibodies and buffers for the ELISA were obtained from BethylLaboratories (Montgomery, Tex.). Human hemoglobin (Hb) was used as theanalyte. The Hb-specific capture antibody (sheep anti-human Hb, 10micrograms per milliliter, in 50 mM Na₂CO₃, pH 9.6) was added throughreservoirs 403 and 404 to coat fluid path 20. A solution of bovine serumalbumin (BSA, 1% in 50 mM Tris, 150 mM NaCl, pH 8) was flowed into fluidpath 20 d for one hour to block the surface and to prevent non-specificadsorption of proteins in subsequent steps. After the sample wasintroduced through fluid path 20 d, a 1 second pulse of 1 kV was used tocreate an opening in closing member 30 d between fluid path 220connected to reservoir 401 and fluid path 20. Anti-Hb antibodyconjugated to alkaline phosphatase (sheep anti-human Hb, 10 microgramsper milliliter in 1% BSA, 50 mM Tris, 150 mM NaCl, pH 8) was pumped intofluid path 20 d with a syringe pump for one hour. Fluids were pumped ata constant rate of 50 microliters per hour. Finally, an opening wasformed in closing member 30 d between fluid path 20 d connected toreservoir 402 and fluid path 220 with a 1 second pulse of 1 kV. Thesubstrate, ELF-97 (0.5 mM in 1% BSA, 50 mM Tris, 150 mM NaCl, pH 8,Molecular Probes, Eugene, Oreg.), was pumped into the device for 10minutes. ELF-97 was soluble and non-fluorescent when phosphorylated.Between each step, fluid path 20 d was rinsed with two aliquots of 50microliter of 50 mM Tris, 100 mM NaCl, 0.05% Tween® 20 (ICI America,North Little Rock, Ak.), pH 8. Upon hydrolysis of the phosphate group byalkaline phosphatase, however, the species precipitated and fluorescedgreen upon exposure to UV light. If no Hb was present in the sample, thesubstrate did not react, and no fluorescence was observed, asillustrated in FIG. 39. If Hb was present, the substrate reacted, and,after about 5 minutes, fluorescence was observed in the device as shownin FIG. 40. Although fluorescent microscopy was used to document theresults, the results were able to be seen by the naked eye with a UVlamp. This experiment demonstrates that a relatively complex assayingdevice may be created using the one way valves of the present invention.

[0135] It will be understood that each of the elements described herein,or two or more together, may be modified or may also find utility inother applications differing from those described above. Whileparticular embodiments of the invention have been illustrated anddescribed, the present invention is not intended to be limited to thedetails shown, since various modifications and substitutions may be madewithout departing in any way from the spirit of the present invention asdefined by the following claims.

What is claimed is:
 1. A microfluidic system comprising: a fluid path;an inlet to the fluid path; an outlet to the fluid path; and a firstclosing member disposed along the fluid path between the inlet and theoutlet; wherein the fluid path has a cross-sectional dimension of lessthan about 500 μm.
 2. The microfluidic system of claim 1, wherein thefirst closing member comprises a first flexible member.
 3. Themicrofluidic system of claim 2, wherein the first closing member has anopen position and a closed position and is constructed and arranged totravel from the closed position to the open position without slidingagainst any portion of the microfluidic system.
 4. The microfluidicsystem of claim 2, wherein the first flexible member comprises a flap.5. The microfluidic system of claim 4, wherein the flap is generallysemicircular in shape.
 6. The microfluidic system of claim 2, whereinthe first flexible member comprises a diaphragm.
 7. The microfluidicsystem of claim 6, wherein the diaphragm comprises an opening.
 8. Themicrofluidic system of claim 7, further comprising a seat constructedand arranged to support the diaphragm around at least the periphery ofthe opening.
 9. The microfluidic system of claim 2, wherein the firstflexible member comprises an elastomer.
 10. The microfluidic system ofclaim 1, wherein the first closing member comprises a firstfree-floating member.
 11. The microfluidic system of claim 10, whereinthe first free-floating member comprises a spherical body.
 12. Themicrofluidic system of claim 1, wherein the fluid path is constructed ofa flexible material.
 13. The microfluidic system of claim 12, whereinthe fluid path is constructed of an elastomer.
 14. The microfluidicsystem of claim 12, further comprising: a flexible support connected tothe flexible material.
 15. The microfluidic system of claim 1, furthercomprising: a second closing member disposed along the fluid pathbetween the inlet and the outlet; and a reservoir disposed along thefluid path between the first closing member and the second closingmember.
 16. The microfluidic system of claim 15, wherein a volume of thereservoir is variable.
 17. The microfluidic system of claim 16, whereinthe reservoir comprises an elastomer.
 18. The microfluidic system ofclaim 1, wherein the fluid path has a cross-sectional dimension of lessthan about 300 μm.
 19. The microfluidic system of claim 18, wherein thefluid path has a cross-sectional dimension of less than about 100 μm.20. The microfluidic system of claim 19, wherein the fluid path has across-sectional dimension of less than about 50 μm.
 21. The microfluidicsystem of claim 1, wherein the closing member comprises a portion of acheck valve.
 22. The microfluidic system of claim 1, wherein the closingmember comprises a substantially sealed barrier and further comprisingan electrode electrically connected to at least one of the inlet of thefluid path and the outlet of the fluid path.
 23. A valve having an openposition and closed position, comprising: a fluid path; an inlet to thefluid path; an outlet to the fluid path; a flexible diaphragm disposedalong the fluid path between the inlet and the outlet; an opening in theflexible diaphragm; and a seat constructed and arranged such that, whenthe valve is in the closed position, the seat obstructs the opening andsupports the flexible diaphragm around at least the periphery of theopening.
 24. The valve of claim 23, wherein the fluid path has across-sectional dimension of less than about 500 μm.
 25. The valve ofclaim 23, wherein the fluid path is constructed of a flexible material.26. The valve of claim 25, wherein the fluid path is constructed of anelastomer.
 27. A microfluidic pump, comprising: a fluid path; an inletto the fluid path; an outlet to the fluid path; a first closing memberdisposed along the fluid path between the inlet and the outlet; a secondclosing member disposed along the fluid path between the inlet and theoutlet; and a reservoir having a variable volume disposed along thefluid path between the first closing member and the second closingmember; wherein the fluid path has a cross-sectional dimension of lessthan about 500 μm.
 28. The microfluidic pump of claim 27, wherein thefluid path is constructed of a flexible material.
 29. The microfluidicpump of claim 28, wherein the fluid path is constructed of an elastomer.30. The microfluidic pump of claim 28, further comprising: a flexiblesupport connected to the flexible material.
 31. A microfluidic system,comprising: a flexible support; a flexible material connected to theflexible support; and at fluid path within the flexible material havinga cross-sectional dimension of less than about 500 μm.
 32. Themicrofluidic system of claim 31, further comprising: an inlet to thefluid path; an outlet to the fluid path; and a first closing memberdisposed along the fluid path between the inlet and the outlet.
 33. Themicrofluidic system of claim 32, further comprising: a second closingmember disposed along the fluid path between the inlet and the outlet;and a reservoir disposed along the fluid path between the first closingmember and the second closing member.
 34. The microfluidic system ofclaim 31, wherein the flexible support is an adhesive tape.
 35. A methodfor making a microfluidic system, comprising: providing a mastercorresponding to the microfluidic system; forming the microfluidicsystem on the master; connecting a support to the microfluidic system;and removing the microfluidic system from the master.
 36. The method ofclaim 35, wherein the connecting step comprises forming a reversibleconnection between the support and the microfluidic system.
 37. Themethod of claim 35, wherein the connecting step comprises adhering thesupport to the microfluidic system.
 38. The method of claim 37, whereinthe support comprises an adhesive tape.
 39. The method of claim 35,wherein the forming step further comprises forming the microfluidicsystem out of a curable elastomer.
 40. The method of claim 35, whereinthe forming step further comprises forming the microfluidic system suchthat it is less than 1 mm thick.
 41. The method of claim 40, wherein theforming step further comprises forming the microfluidic system such thatit is less than 500 μm thick.
 42. The method of claim 41, wherein theforming step further comprises forming the microfluidic system such thatit is less than 100 μm thick.
 43. The method of claim 35, furthercomprising: connecting the microfluidic system to a substrate; andremoving the support from the microfluidic system.
 44. The method ofclaim 43, wherein the removing step comprises: applying a solvent to atleast one of the support and the microfluidic system.
 45. The method ofclaim 44, wherein the step for connecting the microfluidic system to asubstrate is substantially irreversible.
 46. A method for opening amicrofluidic valve, comprising: providing a microfluidic valveincluding: a fluid path, an inlet to the fluid path, an outlet to thefluid path, and a first closing member disposed along the fluid pathbetween the inlet and the outlet, wherein the fluid path has across-sectional dimension of less than about 500 μm; providing a flow ofa fluid through the fluid path; and deflecting the closing member withthe flow from a closed position to an open position without the closingmember sliding against any portion of the microfluidic valve.
 47. Amethod for manipulating a flow of a fluid in a microfluidic system,comprising: providing a fluid path having a cross-sectional dimension ofless that about 500 μm; initiating the flow of the fluid through thefluid path in a first direction; and inhibiting the flow of the fluidthrough the fluid path in a second direction.
 48. The method of claim47, wherein the act of initiating includes urging open a closing memberwith the flow of the fluid in the first direction.
 49. The method ofclaim 47, wherein the act of inhibiting includes urging closed a closingmember with the flow of the fluid in the second direction.
 50. Amicrofluidic system comprising: a first fluid path; a second fluid path;and a first closing member comprised of a voltage degradable materialand disposed between the first and second fluid paths; wherein one ofthe first and second fluid paths has a cross-sectional dimension of lessthan about 500 μm.
 51. The microfluidic system of claim 50, wherein theclosing member comprises a substantially sealed barrier.
 52. Themicrofluidic system of claim 51, wherein the closing member is betweenabout 5 micrometers and about 50 micrometers thick.
 53. The microfluidicsystem of claim 52, wherein the closing member is between about 15micrometers and about 40 micrometers thick.
 54. The microfluidic systemof claim 50, wherein the closing member is constructed of a polymer. 55.The microfluidic system of claim 50, wherein the closing member has abreakdown voltage of less than about 250 volts per micrometer.
 56. Themicrofluidic system of claim 55, wherein the closing member has abreakdown voltage of less than about 150 volts per micrometer.
 57. Themicrofluidic system of claim 56, wherein the closing member has abreakdown voltage of less than about 75 volts per micrometer.
 58. Themicrofluidic system of claim 57, wherein the closing member has abreakdown voltage of less than about 25 volts per micrometer.
 59. Themicrofluidic system of claim 50, further comprising an electrodeelectrically connected to one of the first and second fluid paths. 60.The microfluidic system of claim 59, wherein one of the first and secondfluid paths is connected to an electrical ground.
 61. The microfluidicsystem of claim 59, further comprising an electrical energy sourceconnected to the electrode.
 62. The microfluidic system of claim 61,wherein the electrical energy source comprises a pizoelectricalgenerator.
 63. The microfluidic system of claim 61, wherein theelectrical energy source is sized and adapted to apply a voltage greaterthan the breakdown voltage of closing member.
 64. A microfluidic systemcomprising: a first fluid path; a second fluid path; and a first closingmember comprised of a voltage degradable material and disposed betweenthe first and second fluid paths; wherein the first closing member has athickness of less than about 50 μm.
 65. A microfluidic device,comprising: a substantially sealed fluid reservoir; a fluid positionedwithin the fluid reservoir; a fluid path separated from the fluidreservoir by a closing member; a first electrode connected to the fluidreservoir; and a second electrode connected to the fluid path.
 66. Amethod of manipulating fluid flow in a fluidic system, comprising:creating a voltage difference between a first fluid path and a secondfluid path separated by a closing member, the voltage being sufficientto form an opening in the closing member; and allowing a fluid to flowbetween the first and second fluid paths.
 67. The method of claim 66,wherein creating a voltage difference comprises creating a voltagedifference greater than the breakdown voltage of the closing member. 68.The method of claim 66, further comprising electrically connecting oneof the first and second fluid paths to an electrical energy source. 69.The method of claim 66, further comprising electrically connecting oneof the first and second fluid paths to an electrical ground.
 70. Amethod of testing, comprising: introducing a test fluid into a testreservoir; creating a voltage difference between the test reservoir anda reagent reservoir containing a reagent and separated from the testreservoir by a closing member, the voltage difference being sufficientto make an opening in the closing member; allowing at least one of thetest fluid and the reagent to flow between the test reservoir and thereagent reservoir.
 71. A method of making an opening in a fluidicsystem, comprising: creating a voltage difference between a first fluidpath and a second fluid path separated from the first fluid path by aclosing member sufficient to make an opening in the closing member.